Eur. J. Entomol. 96: 165-168, 1999 ISSN 1210-5759

Chill injury at alternating temperaturesOrchesella in cincta (Collembola: Entomobryidae) andPyrrhocoris apterus (Heteroptera: Pyrrhocoridae)

Z deněk HANČ1 and O ldřich NEDVĚD1,2*

'Faculty of Biological Sciences, University of South Bohemia and institute of Entomology, Academy of Sciences; Branišovská 31, 370 05 České Budějovice, Czech Republic

Key words. Cold hardiness, chill injury, low temperature, survival, mortality, Collembola, Orchesella cincta, Heteroptera, Pyrrhocoris apterus

Abstract. Survival and Lt50 after exposures at constant low temperature were compared to the values obtained at alternating tem­ peratures in two active (summer acclimated) temperate terrestrial arthropods. The experimental regimes used interruptions - daily transfers from the lower temperature to various higher temperatures for two hours or to one high temperature for various durations. In both species the alternating conditions improved survival, implying reparation of the chill injury. In the collembolan Orchesella cincta, there was a maximum Lt50 when the higher exposure temperature was equal to the temperature of rearing (19°C). In the bug Pyrrhocoris apterus, Lt50 increased strongly with increasing higher temperature from 0 to 15°C, and was subsequently constant over the entire physiological range suitable for development (to 35°C). Exposure at 0°C was harmful if continuously applied, but survival increased, relative to a constant exposure at -5°C, if the temperature alternated between -5 and 0°C.

INTRODUCTION + 10°C, 8 h) compared to constant -5°C was found in Laboratory experiments investigating the cold hardiness nymphalid butterfly lnachis io, and no difference was of terrestrial arthropods are usually performed at constant found in Aglais urticae (Pullin & Bale, 1989). In the col­ low temperatures, although in nature the animals are ex­ lembolan Orchesella cincta, an increased duration of the posed to cyclical temperature fluctuations. The impor­ exposure at the higher temperature (0.25 to 2 h per day) tance of measuring survival in insects at temperatures or higher temperatures (+5 to +19°C) of constant duration fluctuating below and above zero, as happens in mild (2 h) had an increasingly positive effect on survival temperate winters, has been stressed by Bale (1989, (Nedvěd et al., 1998). 1991). Similar to the effect of alternating temperatures is the We can hypothesize several different effects on hetero- effect of post exposure conditions. It has been demon­ thermic animals that may be cáused by the fluctuation or strated that higher post exposure temperatures result in alternation between two temperatures. Let us set aside the higher survival than lower ones. Tumock & Bodnaryk freeze tolerant species in which the temperature fluctua­ (1991) reported positive effects on survival of high tions may cause freeze-thawing cycles. In freeze suscepti­ (>15°C) temperature exposure following cold stress (3 ble species, the lower temperature (above SCP) causes days at -15°C) in comparison to a lower (<10°C) post­ chill injury (Nedvěd et al., 1998), but may also serve as a stress temperature in moth Mamestra configurata. When signal for triggering increase in cold hardiness. The syn­ exposure at -15°C for 3 days was followed by exposure thesis of stress proteins is induced by low temperature but at 0°C for various lengths of time before incubation at occurs only after return to higher temperature (Joplin & 20°C, survival decreased linearly with the time spent at Denlinger, 1990; Yocum et al., 1991). At the higher tem­ 0°C, although long exposure at 0°C only caused no mor­ perature some chill injury may be repaired (Chen & Den­ tality. linger, 1992; Nedvěd et al., 1998; Tumock & Bodnaryk, A method to quantify cold hardiness in freeze suscepti­ 1991). The higher temperature may allow physiological ble terrestrial arthropods recently proposed by Nedvěd processes of cold hardening that is cued by the low tem­ (1998) is based on summation of degree days of the cold perature, but requires the higher temperature for effective exposure. One of the two parameters calculated from the expression. Finally, the higher temperature may deaccli- survival data, sum of injurious temperatures (SIT, DD or mate / activate an overwintering animal, which would K.d), indicates the dose of injurious chilling, i.e. product subsequently suffer more injury at the lower temperature. of exposure duration and the temperature difference be­ In the weevil Rhynchaenus fagi, survival i.e. lethal time tween the exposure temperature and the upper limit of the was much longer at cycling temperatures (-15°C and cold injury zone (ULCIZ, °C) that kills a half of a given +2°C cycling over 48 h) than at a constant low tempera­ sample. The model successfully fitted several sets of data ture (-15°C) (Coulson & Bale, 1996). In contrast, a slight on insect survival, from the literature and from original negative effect of alternating temperatures (~5°C, 16 h / measurements. However, all the data were measurements at constant temperatures. This study examines the use of

* Author for correspondence; e-mail: [email protected] .

165 the degree day concept to quantify the amount of chill in­ RESULTS jury under regimes of two alternating temperatures in two Cold hardiness parameters summer-acclimated arthropods. The fifth instar larvae of P. apterus, from long day con­ MATERIAL AND METHODS ditions, supercooled on average to -8°C. The upper limit The modelling was performed on survival data of the collem- of cold injury zone was calculated as +4.2°C, and the sum bolan Orchesella cincta (L.) as published previously (see of injurious temperatures was 34 K.d (data after Hanc, Nedvěd et al., 1998, and results hereafter for rearing and experi­ 1998). mental conditions), and on new data of a pyrrhocorid bug. The supercooling point in adult O. cincta, from long Rearing and exposure conditions day conditions ranged from -5 to -11°C (according to The bug Pyrrhocoris apterus (L.) was reared under long day van der Woude, 1987). ULCIZ calculated according to conditions (18L : 6D, 25°C). Larvae 3-4 days after ecdysis into survival data from Nedved et al. (1998) was +1.4°C, SIT the final (fifth) instar were used in experiments without any low about 14 K.d. temperature acclimation. We measured the supercooling points of 16 individuals to­ Improvement of survivalP. in apterus gether in a single apparatus (Brunnhofer et al., 1991; Nedvěd et Continuous exposure at -5°C caused 50% mortality af­ al., 1995) using cooling rate 10 K/h. Observation of survival af­ ter 3.4 days. When repeated transfer to 25°C was applied, ter exposure at a series of constant low temperatures and various survival improved markedly, even when the duration of durations of exposure (Hanč, 1998) allowed the calculation of such an interruption was only 0.5 h. The longer the dura­ the upper limit of the cold injury zone (ULCIZ) and sum of inju­ tion up to 4 h, the longer the resulting Lt5o (up to 9.6 days, rious temperatures (SIT, according to Nedvěd, 1998). Groups of 20 larvae per vial were transferred to a low tem­ Figs 1, 2). perature cabinet at a constant -5°C. The exposure was inter­ Survival was also much improved by the repeated rupted daily by transfer to a higher constant temperature. In the transfer to various temperatures for two hours, even when first experiment, we used a 2 h exposure at 0, 3, 10, 15, 25, and the temperature was relatively low. The higher the tem­ 35°C. In the second experiment, we used 25°C as the higher perature, up to 35°C, the longer the resulting Lt5o (up to constant temperature, and various exposure duration: 0.5, 1, 2, 8.7 days, Figs 3, 4). 3, and 4 h. One group per treatment was removed at daily intervals, and Improvement of survivalO. in cincta survival assessed as the number of walking larvae after an hour In summer acclimated (19°C, 16L : 8D) adult O. cincta, of recover at 25°C. the Ltso was 3.1 days at a constant -3°C. Survival in­ Calculations of the lethal time creased (up to 10 days) when the exposure was inter­ We used the logistic equation to describe the sigmoid curve of rupted for 0.5, 1, 2, and 4 h at 19°C (Fig. 5), but remained the decrease of survival proportion (5j as a function of time ( t): unchanged (3.18 days) if the interruption lasted only 0.25 „a+b-t h. The longest interruption period (4 h) helped slightly C = —------1 + e a+é'ř less than the nearest shorter period (2 h). The parameters a and b characterize the shape of the curve, The Lt5o was longer than in a constant low temperature with the ratio -a/b equal to the lethal time for 50% of the sam­ when the exposure was interrupted for 2 h at the higher ple (Ltso). The exposure time in all treatments was recalculated temperatures used (Fig. 6). The highest interruption tem­ to include only the time spent at the lower temperature (-5°C), perature (30°C) was much less effective in increasing sur­ e.g. exposure periods with 2 h interruptions were 22, 44, 66 h, vival than the temperature at which the insects were etc. reared.

Fig. 1. Survival of Pyrrhocoris apterus at constant -5°C (•) Fig. 2. Prolongation of Ltso in Pyrrhocoris apterus by transfer and at alternating temperatures -5 / +25°C with various duration from the low temperature (-5°C) to 25°C for various periods of of the higher temperature: 0.5 h (□), 1 h (O), 2 h (A), 3 h (O), time. The curve plotted by least squares smoothing. and 4 h (+). The curves ordered respectively; plotted by logistic regression.

166 Fig. 3. Survival of Pyrrhocoris apterus at constant -5°C (•) Fig. 4. Prolongation of Lt50 in Pyrrhocoris apterus by transfer and at alternating temperatures from -5 to 0°C (□), 3°C (O), from the low temperature exposure (-5°C) to various tempera­ 10°C (A), 15°C (O), 25°C (+), and 35°C (♦). The curves or­ tures for 2 h. The curve plotted by least squares smoothing. dered respectively; plotted by logistic regression.

DISCUSSION quent survival at low temperature will decrease, as in the butterfly. Direction of the effect of high temperature pulses In both species, survival was higher (Ltso longer) when Effect of various pulse durations the exposure at low temperature was interrupted every The positive effect of alternating temperatures in­ day for a short period by transfer to a higher temperature. creased with the duration of the interruption of the cold All combinations of duration and temperature had a posi­ exposure. However, beyond certain rather short periods, tive effect, thus agreeing with results on the weevil Rhyn- the improvement became constant, i.e. extension of the chaenus fagi (Coulson & Bale, 1996), but contrasting high temperature pulse produced no further increase in with the butterfly Inachis io(Pullin & Bale, 1989). survival. Possibly, the switch from the increasing to the There is a great physiological difference between the steady improvement (here about 1 h, see Fig. 2) might be arthropods investigated in this paper and the two studied shifted to longer durations if lower temperatures were earlier. Both the bug and the collembolan were taken used. In natural fluctuating conditions, the duration of the from summer conditions - long day and high temperature warm period during the day may last several hours, but - and were active, not cold acclimated. In such cases it is the highest temperature of the day in the insect hiber- not surprising that high temperatures decreased mortality nacula may be lower than in our experiments when ani­ caused by chilling through reparation of the cold injury mals were not acclimated to cold. (Nedved et al., 1998; Tumock & Bodnaryk, 1991). The Effect of various high temperatures weevil and the butterfly were overwintering, cold accli­ Although the effect of alternating temperatures was mated. In such samples we may expect two opposite reac­ positive in both species, there was an interspecific differ­ tions. If the higher of the two alternating temperatures is ence (Figs 4, 6). In O. cincta, the effect was weak at not too high, just within the range of temperatures that the lower temperatures and increased almost exponentially overwintering insect may encounter, repair of injury may with increasing temperature. Very high temperature was occur and hence a reduction in mortality, as was the case either less effective in repair of the cold injury, or it re­ in the weevil. If the higher temperature is too high, it may paired cold injury but also caused some heat injury. cause deacclimation as occurs in spring, and the subse­

interruption duration (hours) Fig. 5. Prolongation of Lt50 in Orchesella cincta by transfer Fig. 6. Prolongation of Lt50 in Orchesella cincta by transfer from the low temperature exposure (-3°C) to 19°C for various from the low temperature exposure (-3°C) to various tempera­ periods of time. The curve plotted by least squares smoothing. tures for 2 h. The curve plotted by least squares smoothing.

167 In P. apterus, the increase in survival was quite high ACKNOWLEDGEMENTS. The study was supported by the grant even at low temperatures, it increased at moderate tem­ No. 206/97/0619 of the Grant Agency of the Czech Republic. peratures and was constant over the entire physiological REFERENCES range suitable for development (LDT 15°C - UDT 35°C, Nováková & Nedvěd, 1999). The higher lethal tempera­ Bale J.S. 1989: Cold hardiness and overwintering of insects. ture (38°C) was not used in the experiments. Whether the Agrie. Zool. Rev. 3: 157-192. switch from increasing to constant part of the curve on Bale J.S. 1991: Implications of cold hardiness for pest manage­ ment. In Lee R.E. Jr. & Denlinger D.L. (eds): Insects at Low Fig. 4 would be also close to LDT if a shorter duration of Temperature. 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H anč Z. 1998: Survival o f Insects at Low Temperatures, Non- A different pattern of improvement of survival by a Freeze Mortality and Effect o f Low Temperature Exposures high temperature pulse was found in Sarcophaga crassi- Interrupted by Higher Temperatures. MSc. thesis, University palpis (see Chen & Denlinger, 1992): interruption of cold of South Bohemia, České Budějovice, 35 pp. (in Czech). exposure had a positive effect on survival of subsequent Joplin K.H. & D enlinger D.L. 1990: Developmental and tissue cold shock when the insects were exposed at +15°C. Nei­ specific control of the heat shock induced 70kDa related pro­ ther lower (5, 10) nor higher (20, 25, 30°C) temperatures teins in the flesh fly, Sarcophaga crassipalpis. J. Insect had the protective effect. Physiol. 36:239-249. Nedvěd O. 1998: Modelling the relationship between cold injury Helpful injurious temperatures and accumulated degree days in terrestrial arthropods. Cryo- An interesting phenomenon becomes apparent when the Letters 19:267-274. improvement of survival by various high temperatures is Nedvěd O., H odková M., Brunniiofer V. & H odek 1. 1995: Si­ considered in relation to the upper limit of the cold injury multaneous measurement of low temperature survival and su­ zone. The data on both species can be recalculated in such percooling in a sample of insects. Cryo-Letters 16: 108-113. a way that only the duration of exposure at the lower of Nedvěd O., Lávy D. & V erhoef H.A. 1998: Modelling time- temperature relationship in cold injury and effect of high tem­ the two alternating temperatures is cumulated. Hence, if perature interruptions on survival in a chill sensitive the higher of the two temperatures has no effect on the collembolan. Fund. Ecol. 12:816-824. animals - neither injurious nor reparative - the resulting Nováková J. & Nedvěd O. 1999: Developmental time and body Lt5o should be the same as in the constant exposure. It mass in Pyrrhocoris apterus reared at constant and alternating seems to be true when the temperatures are close to UL­ temperatures. Entomol. Problems 30 (in press). CIZ (1°C) in O. cincta. However, temperatures well bel­ P ullin A.S. & Bale J.S. 1989: Effects of low temperature on ow the ULCIZ (4°C) in P. apterus had a clearly positive diapausing Agíais urticae and Inachis io (Lepidoptera: Nym- effect on survival. This phenomenon makes it impossible phalidae): cold hardiness and overwintering survival. J. Insect to summarize chill injury in this species in alternating Physiol. 35: 277-281. T urnock W.J. & Bodnaryk R.P. 1991: Latent cold injury and temperatures according to a degree-day concept. The pa­ its conditional expression in the bertha armyworm, Mamestra rameter SIT (Nedvěd, 1998) is valid only in experiments conflgurata (Noctuidae: Lepidoptera). Cryo-Letters 12: with constant temperatures. 377-384. However, we do not know, whether in the overwinter­ van der W oude H.A. 1987: Seasonal changes in cold hardiness ing, cold-acclimated animals, the chill injury is simply cu­ of temperate Collembola. Oikos 50: 231-238. mulative or if there is a repair of injury similar to the Y ocum G.D., J oplin K.H. & D enlinger D.L. 1991: Expression situation in P. apterus. The present results might be use­ of heat shock proteins in response to high and low tempera­ fully employed in designing regimes for the long-term ture extremes in diapausing pharate larvae of the gypsy moth, cold storage of active insects for laboratory experiments Lymantria dispar. Arch. Insect Biochem. Physiol. 18: or for the timed release of biological control agents. 239-249. Received September 28, 1998; accepted January 15, 1999

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